U.S. patent application number 09/124280 was filed with the patent office on 2002-03-21 for vaccine for prevention of gram-negative bacterial infections and endotoxin related diseases.
Invention is credited to PORRO, MASSIMO.
Application Number | 20020034520 09/124280 |
Document ID | / |
Family ID | 22413904 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034520 |
Kind Code |
A1 |
PORRO, MASSIMO |
March 21, 2002 |
VACCINE FOR PREVENTION OF GRAM-NEGATIVE BACTERIAL INFECTIONS AND
ENDOTOXIN RELATED DISEASES
Abstract
A vaccine is disclosed which is useful for protecting a host
from Gram negative infections and the effects of endotoxin,
therefore preventing sepsis and septic shock. The vaccine is
prepared by combining LPS free or in conjugate form with a
stoichiometric excess of a peptide of the formula: (a) (A).sub.n
wherein A is Lysine or Arginine and n is an integer with a minimum
value of 7; (b) (AB).sub.m wherein A is Lysine or Arginine and B is
a hydrophobic amino acid selected from the group consisting of
Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; m is an integer with a minimum value of 3; and (c)
(ABC).sub.p wherein A is a cationic amino acid which is Lysine or
Arginine; B and C are hydrophobic amino acids which may be the same
or different and are selected from the group consisting of Valine,
Leucine, Isoleucine, Tyrosine, Phenylalanine and Tryptophan; p is
an integer with a minimum value of 2.
Inventors: |
PORRO, MASSIMO; (SIENA,
IT) |
Correspondence
Address: |
JAMES V COSTIGAN
HEDMAN GIBSON & COSTIGAN
1185 AVENUE OF THE AMERICAS
NEW YORK
NY
100362601
|
Family ID: |
22413904 |
Appl. No.: |
09/124280 |
Filed: |
July 29, 1998 |
Current U.S.
Class: |
424/234.1 |
Current CPC
Class: |
A61K 39/385 20130101;
Y02A 50/30 20180101; A61K 2039/6081 20130101; A61K 39/0275
20130101; A61K 2039/6037 20130101; A61K 2039/55572 20130101; Y02A
50/482 20180101; A61K 39/095 20130101; A61P 31/04 20180101; Y02A
50/484 20180101 |
Class at
Publication: |
424/234.1 |
International
Class: |
A61K 039/02 |
Claims
I claim:
1. A vaccine for preventing gram negative infections and the
effects of endotoxins which comprises a complex obtained by
combining LPS free or in conjugate form with a stoichiometric
excess of a peptide of the formula: (a) (A).sub.n wherein A is
Lysine or Arginine and n is an integer with a minimum value of 7;
(b) (AB).sub.m wherein A is Lysine or Arginine and B is a
hydrophobic amino acid selected from the group consisting of
Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; m is an integer with a minimum value of 3; and (c)
(ABC).sub.p wherein A is a cationic amino acid which is Lysine or
Arginine; B and C are hydrophobic amino acids which may be the same
or different and are selected from the group consisting of Valine,
Leucine, Isoleucine, Tyrosine, Phenylalanine and Tryptophan; p is
an integer with a minimum value of 2.
2. A vaccine as defined in claim 1 wherein the peptide is a linear
or cyclic peptides having units of the formula: (a) (A).sub.n
wherein A is Lysine or Arginine and n is an integer with a value of
7 to 16; (b) (AB).sub.m wherein A is Lysine or Arginine and B is a
hydrophobic amino acid selected from the group consisting of
Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; m is an integer with a value of 4 to 20; and (c)
(ABC).sub.p wherein A is a cationic amino acid which is Lysine or
Arginine; B and C are hydrophobic amino acids which may be the same
or different and are selected from the group consisting of Valine,
Leucine, Isoleucine, Tyrosine, Phenylalanine and Tryptophan; p is
an integer with a value of 4 to 20.
3. A vaccine as defined in claim 1 wherein the LPS is derived from
N. menigitidis.
4. A vaccine as defined in claim 1 wherein the LPS is derived from
Salmonella typhi.
5. A vaccine as defined in claim 1 where the amount of peptide is
from 2-10 to 2-5000 times the weight of the LPS.
6. A vaccine as defined in claim 1 wherein the peptide has units of
the formula (AB).sub.m.
7. A vaccine as defined in claim 1 wherein the peptide has units of
the formula (ABC).sub.p.
8. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys).sub.10. (SEQ ID NO: 1)
9. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Glu).sub.5. (SEQ ID NO: 4)
10. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Phe).sub.5. (SEQ ID NO: 5)
11. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Phe-Leu-Lys-Lys-Thr-Leu. (SEQ ID NO: 6)
12. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Phe-Leu).sub.2-Lys. (SEQ ID NO: 7)
13. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Phe-Leu).sub.3-Lys. (SEQ ID NO: 8)
14. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Arg-Tyr-Val).sub.3. (SEQ ID NO: 9)
15. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Phe-Phe).sub.3-Lys. (Seq ID NO: 10)
16. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys-Leu-Leu).sub.3 (SEQ ID NO: 11)
17. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys).sub.6(Phe-Lys).sub.2. (SEQ ID NO: 12)
18. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
5 Cys-(Lys).sub.5-Cys (SEQ ID NO: 13) s-----------s.
19. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
6 Cys-Lys-Phe-Lys-Lys-Cys (SEQ ID NO: 14)
s--------------------s.
20. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
7 Lys-Phe-Lys-Cys-Lys-Phe-Lys-Phe-Lys-Cys (SEQ ID NO: 15)
s-----------------------s.
21. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
8 Lys-Leu-Lys-Cys-Lys-Leu-Lys-Leu-Lys-Cys (SEQ ID NO: 16)
s------------------------s.
22. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
9 Arg-Thr-Arg-Cys-Arg-Phe-Lys-Arg-Arg-Cys (SEQ ID NO: 17)
s------------------------s.
23. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
10 Lys-Cys-(Lys-Phe-Lys).sub.2-Cys-Lys (SEQ ID NO: 18)
s-------------------s.
24. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
11 Cys-(Lys).sub.4-(Phe).sub.4-Cys (SEQ ID NO: 19)
s------------------s.
25. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
12 Cys-(Lys-Phe-Leu).sub.3-Lys-Cys (SEQ ID NO: 20)
s-----------------------s.
26. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Val-Lys-Ala-Leu-Arg-Val-Arg-Arg-Leu (SEQ ID NO: 21)
27. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Ser-Leu-Ser-Leu-Lys-Arg-Leu-Thr-Tyr-Arg (SEQ ID
NO:22)
28. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-Val (SEQ ID NO: 23)
29. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Phe-Leu-Lys-Pro-Gly-Lys-Val-Lys-Val (SEQ ID NO: 24)
30. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Asp-Leu-Lys-Arg-Ile-Lys-Ile (SEQ ID NO: 25)
31. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Trp-Lys-Ala-Gln-Lys-Arg-Phe-Leu (SEQ ID NO: 26)
32. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Trp-Lys-Ala-Gln-Lys-Arg-Phe-Leu-Lys (SEQ ID NO:
27)
33. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Arg-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe (SEQ ID
NO:28)
34. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
13 Cys-Gln-Trp-Lys-Ser-Ser-Asp-Ile-Arg-Cys-Gly-Lys (SEQ ID NO: 29)
s------------------------------------s
35. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
14 Cys-Lys-Phe-Leu-Lys-Lys-Cys (Seq ID NO: 30)
s------------------------s
36. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
15 Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys (SEQ ID NO: 31) s - - -
- - - - - - - - s
37. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Phe-Leu-Lys-Lys-Thr(SEQ ID NO: 32)
38. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
16 Cys-Lys-Lys-Leu-Phe-Lys-Cys-Lys-Thr-Lys (SEQ ID NO: 33) s - - -
- - - - - - - - s
39. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
17 Cys-Lys-Lys-Leu-Phe-Lys-Cys-Lys-Thr (SEQ ID NO: 34) s - - - - -
- - - - - - s
40. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
18 Ile-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys (SEQ ID NO: 35) s -
- - - - - - - - - - s
41. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Ile-Lys-Thr-Lys-Lys-Phe-Leu-Lys-Lys-Thr(SEQ ID NO: 36)
42. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Ile-Lys-Phe-Leu-Lys-Phe-Leu-Lys-Phe-Leu-Lys(SEQ ID NO:
37)
43. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Phe-Leu-Lys-Phe-Leu-Lys(SEQ ID NO: 38)
44. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Arg-Tyr-Val-Arg-Tyr-Val-Arg-Tyr-Val(SEQ ID NO: 39)
45. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Phe-Phe-Lys-Phe-Phe-Lys-Phe-Cys(SEQ ID NO: 40)
46. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Ile-Lys-Phe-Leu-Lys-Phe-Leu-Lys-Phe-Leu(SEQ ID NO:41)
47. A vaccine as defined in claim 1 wherein the peptide is of the
formula: (Lys).sub.6Phe-Leu-Phe-Leu(SEQ ID NO:42)
48. A vaccine as defined in claim 1 wherein the peptide is of the
formula:
19 Cys-Lys-Phe-Lys-Phe-Lys-Phe-Lys-Phe-Cys (SEQ ID NO: 43
s------------------------------------s
49. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Trp-Lys-Ala-Gln-Lys-Arg-Phe-Leu-Lys(SEQ ID NO: 44)
50. A vaccine as defined in claim 1 wherein the peptide is of the
formula: Lys-Arg-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe(SEQ ID NO:
45)
51. A method for the preparation of a vaccine for prevention of
gram-negative infections and the effects of endotoxins, said method
comprising combining LPS with a stoichiometric excess of a peptide
of the formula: (a) (A).sub.n wherein A is Lysine or Arginine and n
is an integer with a minimum value of 7; (b) (A).sub.m wherein A is
Lysine or Arginine and B is a hydrophobic amino acid selected from
the group consisting of Valine, Leucine, Isoleucine, Tyrosine,
Phenylalanine and Tryptophan; m is an integer with a minimum value
of 3; and (c) (ABC).sub.p wherein A is a cationic amino acid which
is Lysine or Arginine; B and C are hydrophobic amino acids which
may be the same or different and are selected from the group
consisting of Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine
and Tryptophan; p is an integer with a minimum value of 2.
52. A vaccine as defined in claim 1 which is combined or
administered with other vaccine components.
53. A vaccine as defined in claim 1 which contains an LPS peptide
complex derived from more than one species of bacteria.
Description
[0001] The present invention is concerned with providing a vaccine
for prevention of bacterial infections caused by gram-negative
bacteria and for the prevention of the biological effects of
homologous endotoxins.
BACKGROUND OF THE INVENTION
[0002] LPS is the major antigen of gram-negative bacteria. This
material is a glycophospholipid consisting of an antigenic,
variable size, carbohydrate chain covalently linked to lipid A, the
conserved hydrophobic region structurally defined as N,O-acyl
beta-1,6-D-glucosamine 1,4'-bisphosphate. Toxicity of LPS is
expressed by lipid A through the interaction with B-cells and
macrophages of the mammalian immune system, a process leading to
the secretion of proinflammatory cytokines, mainly TNF, which may
have fatal consequences for the host. Lipid A also activates human
T-lymphocytes (Th-1) "in vitro" as well as murine CD4+ and CD8+
T-cell "in vivo", a property which allows the host's immune system
to mount a specific, anamnestic IgG antibody response to the
variable-size carbohydrate chain of LPS. On these bases, LPS has
been recently recognized as a T-cell dependent antigen "in
vivo".
[0003] In order to fully express toxicity, LPS must retain its
supramolecular architecture, through the association of several
units of glycophospholipid monomers forming the lipid A structure.
This conformational rearrangement of the molecule is also
fundamental for full expression of the immunogenic characteristic.
Therefore, dissociation of these intrinsic properties of the
molecule appear to be of crucial interest for proposing the design
of LPS-based vaccines related to the prophylaxis of acute and
chronic pathologies due to gram-negative bacterial infections like
meningococcal meningitis, typhoid fever and Helicobacter
pylori-induced gastritis.
[0004] Sepsis and septic shock are well defined clinical conditions
that are caused by bacteria and by LPS which is the endotoxin
elaborated by the bacteria responsible for the above mentioned
pathologies. The present inventor has described treatment regimens
for septic shock which are based on the use of a defined class of
peptides that have been demonstrated to be capable of neutralizing
LPS in vivo and protecting mammal from septic shock induced by
LPS.
[0005] The treatment of a subject for septic shock requires that
the subject who has symptoms of LPS toxicity be given the peptide
when symptoms appear. The peptide is not an immunogenic compound
for the production of antibodies to LPS and it use prophylactically
will not prevent sepsis which is caused by the bacteria which
release LPS. LPS has immunogenic properties but it is too toxic to
be used to induce the production of antibodies in a host who is to
be protected from the effects of a bacterial infection and from the
effects of LPS which is released by certain bacterial
infections.
[0006] Certain of the peptides are disclosed in U.S. Pat. No.
5,371,186, and information about the basis of the vaccine is
disclosed in J. Endotoxin Res. (1997) 4(4)261-272, which is
incorporated by reference.
SUMMARY OF THE INVENTION
[0007] The applicant has discovered that a vaccine may be prepared
by making an endotoxoid that is made by combining LPS free or in
conjugate form with a stoichiometric excess of a peptide of the
formula:
[0008] (a) (A).sub.n wherein A is Lysine or Arginine and n is an
integer with a minimum value of 7;
[0009] (b) (AB).sub.m wherein A is Lysine or Arginine and B is a
hydrophobic amino acid selected from the group consisting of
Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; m is an integer with a minimum value of 3; and
[0010] (c) (ABC).sub.p wherein A is a cationic amino acid which is
Lysine or Arginine; B and C are hydrophobic amino acids which may
be the same or different and are selected from the group consisting
of Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; p is an integer with a minimum value of 2. The peptides
of the invention may be terminated independently with a hydrogen
atom or any of the naturally occurring amino acids, a fatty acid
residue or a carbohydrate residue.
[0011] The vaccine is particularly useful for the prevention of
gram-negative infections and the effects of endotoxins.
[0012] Accordingly, it is a primary object of the invention to
provide a method for preparing a vaccine for the prevention of
sepsis and septic shock;
[0013] It is also an object of the invention to provide a novel
vaccine for the prevention of sepsis and septic shock.
[0014] It is also an object of the invention to provide a novel
vaccine based on an endotoxoid complex of LPS/peptide or conjugated
LPS/peptide complex derived from homologous LPS.
[0015] It is also an object of this invention to provide novel
compositions and methods for the treatment of microbial
infections.
[0016] These and other objects of the invention will become
apparent from the appended specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is graph which compares the percent of TNF produced
"in vivo" by LPS A1 and various LPS A1 conjugate antigens
detoxified by a peptide which is representative of the class of
peptides which may be used in the present invention.
[0018] FIG. 2a comprises a graph which illustrates the kinetic of
serum IgG production in SW mice induced by LPS A1, using IgG which
is specific for LPS A1. The filled squares are LPS A1; open squares
with the cross are LPS A1/SAEP2 peptide
1 (SAEP2 is: Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys) s - - - - - -
- - - - - s
[0019] complex (ratio LPS A1/SAEP2=1:250(v/v)); inverted open
triangle BSA-LPS A1; filled circle BSA-LPS A1/peptide SAEP2 complex
(ratio LPS A1/SAEP2=1:250 (v/v)). The titers of IgG, represented by
the OD value of each symbol were detected by ELISA in the sera pool
of each animal group at a standard dilution of 1:200(v/v)
[0020] FIG. 2b comprises a graph which is similar to FIG. 2a except
that the serum was specific for the carrier protein BSA.
[0021] FIG. 3 is a graph which illustrates the lack of toxicity by
quantitation of the TNF release in the serum of SW mice 90 min.
after s.c. injections (3 weeks apart), of endotoxoid of N.
meningitidis A1 prepared in different formulations.
[0022] FIG. 4a shows the kinetic of serum IgG production in SW mice
induced by four doses of endotoxoid of N. meningitidis A1 injected
s.c. three weeks apart. All sera were diluted at 1:800(v/v).
[0023] FIG. 4b shows the kinetic of serum IgG production in CD1
mice induced by four doses of endotoxoid of Salmonella typhimurium
injected s.c. three weeks apart. All sera were diluted at
1:10,000(v/v).
[0024] FIG. 5a is a graph which shows the immunochemical
specificity of mouse IgG antibody induced by the endotoxoid A1
prepared from N. meningitidis Strain A1.
[0025] FIG. 5b is a graph which shows the immunochemical
specificity of mouse IgG antibody induced by the endotoxoid Ty
prepared from S.enterica (Serovar typhimurium).
[0026] FIG. 6a is a graph which shows the protection against an
i.p. challenge of S. enterica (Serovar typhimurium) in CD1 mice
immunized either with endotoxoid Ty or homologous/heterologous
antigens as controls where the LD.sub.100 (dose of bacteria killing
100% of the animal population) is 4.times.10.sup.5 cells.
[0027] FIG. 6b is a graph which is the same as FIG. 5a except that
it shows the protection against an i.p. challenge of S. enterica in
CD1 mice immunized either with endotoxoid Ty or
homologous/heterologous antigens as controls where the LD.sub.50 is
9.times.10.sup.3 cells.
DETAILED DESCRIPTION OF THE INVENTION
[0028] The toxic characteristics of LPS may be abrogated without
elimination of the antigenic and immunogenic properties of LPS by
binding the LPS (via the lipid A moiety) to a peptide as defined in
the present application. Typical species of bacteria which produce
LPS include N. meningitidis Group A,B,C. W135, Y; E. coli
(especially strain 0157); Salmonella typhi; Salmonella paratyphi;
(A and B) Shigella flexneri; non-typeable Haemophilus influenzae;
Haemophilus influenzae, type b; Helicobacter pylori; Chlamydia
trachomatis; Chlamydia pneumoniae; Bordatella pertussis; Brucella;
Legionella pneumophia; Vibrio cholera (type 01 and non-01);
Moraxella catharralis; Pseudomonas aeruginosa; and Klebsiella
pneumonia (all species). In particular, the toxicity of
structurally different LPS' has been completely abrogated by a
lipid A-binding cyclic decapeptide, without affecting the
structural integrity of the lipid A moiety and the supramolecular
architecture of the antigen. It has been found that different LPS'
exhibit an active lipid A moiety with a binding site which can be
stoichiometrically saturated in vitro, with high affinity, with a
peptide according to the present invention. However, it has been
found that for in vivo detoxification of LPS with a peptide
according to the present invention, it is necessary to use an
excess of peptide with respect to the stoichiometric amount
required "in vitro" to sufficiently detoxify LPS for preparing an
immunogenic endotoxoid (complex) which will induce antibody
formation without any unacceptable toxicity. It is believed that
the stoichiometric excess is necessary to significantly stabilize
the LPS-peptide complex from the likely antagonistic activity of
natural LPS-receptor proteins present on specialized cells of the
immune system which bear amino acid sequences similar to that of
the peptides used in the present invention.
[0029] Generally, the immunogenic compound, or endotoxoid complex
of the invention, is prepared by combining LPS, derived from a
bacterial source, with from 2-10 to 2-5000, preferably from 10-5000
and especially preferably from 250-2500 times its weight, of a
peptide as described herein based on a weight/weight ratio of LPS
to the peptide. Higher molecular weight peptides may require a
higher ratio of peptide to LPS. It is also possible to first
conjugate the LPS with a protein such as bovine serum albumin,
tetanus toxoid or diphtheria toxoid or non-toxic diphtheria mutant
proteins (CRM197) or outer-membrane proteins (OMP) prior to
combining the LPS with the peptide to form the endotoxoid complex.
Generally a ratio of 2:1 of LPS to BSA may be employed in the
conjugation procedure to yield a covalent conjugate of LPS:BSA=1
(w/w).
[0030] The peptides which may be complexed with the lipid A moiety
of LPS include linear or cyclic peptides having units of the
formula:
[0031] (a) (A).sub.n wherein A is Lysine or Arginine and n is an
integer with a minimum value of 7;
[0032] (b) (AB).sub.m wherein A is Lysine or Arginine and B is a
hydrophobic amino acid selected from the group consisting of
Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; m is an integer with a minimum value of 3; and
[0033] (c) (ABC).sub.p wherein A is a cationic amino acid which is
Lysine or Arginine; B and C are hydrophobic amino acids which may
be the same or different and are selected from the group consisting
of Valine, Leucine, Isoleucine, Tyrosine, Phenylalanine and
Tryptophan; p is an integer with a minimum value of 2. The peptides
of the invention may be terminated independently with a hydrogen
atom or any of the naturally occurring amino acids, a fatty acid
residue or a carbohydrate residue. In addition the retroinverted
peptides, the enantiomer amino acid sequences (all -D amino acids
in the sequence), the diastereomer amino acid sequences (-D and -L
amino acids in the sequence), and the peptide sequences in which
the amino acids are inverted with respect to their original
position in the sequence which are based on the peptides described
herein may also be employed.
[0034] The preferred peptides for use in the invention will also
have a ratio of aliphatic cationic amino acids to hydrophobic amino
acids (R.sub.c/h) of at least 0.5 and within the range of about 0.5
to 10.0 which is computed by using the solvent parameter values
only for those amino acids which are present in the peptides which
have a solvent parameter value equal to or greater than
+1.5kcal/mol (lysine and arginine) and -1.5 kcal/mol (valine,
isoleucine,leucine, tyrosine, phenylalanine and tryptophane) as
measured according to Levitt, J. Mol. Biol. 104,59 (1976), which is
incorporated by reference.
[0035] The peptide sequence for use in preparing a vaccine
according to the invention will preferably comprise six to ten
amino acid residues containing a minimum of three aliphatic
cationic amino acids, with a ratio of aliphatic cationic amino
acids to hydrophobic amino acids of equal to or greater than 0.5
(R.sub.c/h wherein c is the number of cationic amino acids in the
peptide and h is the number of hydrophobic amino acids in the
peptide). This ratio is believed to be the minimum although
sequences of ten amino acids with a ratio (R.sub.c/h) equal to or
greater than 1.0 are optimal for expression of biological
activity.
[0036] The peptide units which are represented by formula (a), (b)
and (c) represent discrete peptides which will also potentiate
antibiotics as well as peptides which will bind and neutralize
endotoxin in the LAL test and which include as a part of their
structure units of formula (a), (b) and (c), in addition to other
amino acids, are included within the peptides which comprise the
invention.
[0037] The preferred minimum values for n, m and p have been
determined experimentally on the basis of the observation that when
the peptide is linear, it should have at least 7 amino acid units
and when said peptide is cyclic or a polymer having several cycles,
i.e. 2 to 6 cycles, it will have a ring structure that has a
minimum of 6 amino acid units; said peptides having a ratio of
aliphatic cationic amino acids to hydrophobic amino acids which is
equal to or greater than 0.5.
[0038] When the peptides are of the formula (A).sub.n, (AB).sub.m
or (ABC).sub.p, i.e. when these formulas do not represent units of
a larger peptides, n will be from 7 to 500 and preferably from 7 to
16; m will be from 3 to 200 and preferably from 4 to 20 and p will
be from 2 to 100 and preferably from 4 to 20.
[0039] Examples of the peptides are listed below.
2 (Lys).sub.10 (SEQ ID NO: 1); (Lys).sub.30 (SEQ ID NO: 2);
(Lys).sub.434 (SEQ ID NO: 3); (Lys-Asp).sub.5 (SEQ ID NO: 4);
(Lys-Phe).sub.5 (SEQ ID NO: 5); Lys-Phe-Leu-Lys-Lys-Thr-Leu (SEQ ID
NO: 6); (Lys-Phe-Leu).sub.2-Lys (SEQ ID NO: 7);
(Lys-Phe-Leu).sub.3-Lys (SEQ ID NO: 8); (Arg-Tyr-Val).sub.3 (SEQ ID
NO: 9); (Lys-Phe-Phe).sub.3-Lys (Seq ID NO: 10);
(Lys-Leu-Leu).sub.3 (SEQ ID NO: 11); (Lys)6(Phe-Lys).sub.2 (SEQ ID
NO: 12); Cys-(Lys).sub.5-Cys (SEQ ID NO: 13); s-----------s
Cys-Lys-Phe-Lys-Lys-Cys (SEQ ID NO: 14); s----------------s
Lys-Phe-Lys-Cys-Lys-Phe-Lys-Phe-Lys-Cys (SEQ ID NO: 15)
s------------------------s Lys-Leu-Lys-Cys-Lys-Leu-Lys-Leu-Lys-Cys
(SEQ ID NO: 16) s------------------------s
Arg-Thr-Arg-Cys-Arg-Phe-Lys-A- rg-Arg-Cys (SEQ ID NO: 17);
s------------------------s Lys-Cys-(Lys-Phe-Lys).sub.2-Cys-Lys (SEQ
ID NO: 18) s-------------------s Cys-(Lys).sub.4-(Phe).sub.4-Cy- s
(SEQ ID NO: 19) s------------------s
Cys-(Lys-Phe-Leu).sub.3-Lys-Cys (SEQ ID NO: 20)
s-----------------------s Val-Lys-Ala-Leu-Arg-Val-Arg-Arg- -Leu
(SEQ ID NO: 21); Lys-Ser-Leu-Ser-Leu-Lys-Arg-Leu-Thr- -Tyr-Arg (SEQ
ID NO: 22); Lys-Val-Arg-Lys-Ser-Phe-Phe-Lys-- Val (SEQ ID NO: 23);
Phe-Leu-Lys-Pro-Gly-Lys-Val-Lys-Val (SEQ ID NO: 24);
Lys-Glu-Leu-Lys-Arg-Ile-Lys-Ile (SEQ ID NO: 25);
Lys-Trp-Lys-Ala-Gln-Lys-ArgPhe-Leu (SEQ ID NO: 26);
Lys-Trp-Lys-Ala-Gln-Lys-Arg-Phe-Leu-Lys (SEQ ID NO: 27);
Lys-Arg-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe (SEQ ID NO: 28); and
Cys-Gln-Ser-Trp-Lys-Ser-Ser-Glu-Ile-Arg-Cys-- Gly-Lys (SEQ ID NO:
29). s----------------------------------------s
Cys-Lys-Phe-Leu-Lys-Lys-Cys (SEQ ID NO: 30) s - - - - - - - - - - -
s Lys-Thr-Lys-Cys-Lys-phe-Leu-Lys-Lys-C- ys (SEQ ID NO: 31) s - - -
- - - - - - - - s Lys-Phe-Leu-Lys-Lys-Thr (SEQ ID NO: 32)
Cys-Lys-Lys-Leu-Phe-Lys-Cys-Lys-Thr-Lys (SEQ ID NO: 33) s - - - - -
- - - - - - s Cys-Lys-Lys-Leu-phe-Lys-Cys-Lys-Thr (SEQ ID NO: 34) s
- - - - - - - - - - - -s
Ile-Lys-Thr-Lys-Cys-Lys-Phe-Leu-Lys-Lys-Cys (SEQ ID NO: 35); s - -
- - - - - - - - - s Ile-Lys-Thr-Lys-Lys-Phe-Leu-Lys-Lys-Thr (SEQ ID
NO: 36) Ile-Lys-Phe-Leu-Lys-Phe-Leu-Lys-Phe-Leu-Lys (SEQ ID NO: 37)
Lys-Phe-Leu-Lys-Phe-Leu-Lys (SEQ ID NO: 38)
Arg-Tyr-Val-Arg-Tyr-Val-Arg-Tyr-Val (SEQ ID NO: 39)
Lys-Phe-Phe-Lys-Phe-Phe-Lys-Phe-Phe (SEQ ID NO: 40)
Ile-Lys-Phe-Leu-Lys-Phe-Leu-Lys-Phe-Leu (SEQ ID NO: 41)
(Lys)6Phe-Leu-Phe-Leu (SEQ ID NO: 42)
Cys-Lys-Phe-Lys-Phe-Lys-Phe-Lys-Phe-Cys (SEQ ID NO: 43);
s------------------------------------s
Lys-Trp-Lys-Ala-Gln-Lys-Arg-Phe-Leu-Lys (SEQ ID NO: 44)
Lys-Arg-Leu-Lys-Trp-Lys-Tyr-Lys-Gly-Lys-Phe (SEQ ID NO: 45)
[0040] The peptides for use in the present invention may be
synthesized by classical methods of peptide chemistry using manual
or automated techniques as well as by DNA recombinant technology.
The synthetic procedure comprises solid phase synthesis by Fmoc
chemistry, cleavage (TFA 95%+Et-(SH).sub.2 5%) followed by vacuum
evaporation. Thereafter, the product is dissolved in 10% acetic
acid, extracted with ether, concentrated at 0.1 mg/ml at pH of
6.0-7.5. Stirring under filtered air followed for 1 to 6 hours in
case of the Cysteine-containing peptides and finally desalting by
reverse phase chromatography is carried out.
[0041] A particular automated method of preparing peptides for use
in the present invention is based on the use of an automatic
synthesizer (Milligen Mod.9050 (MILLIPORE, Burlington, Mass.) on a
solid phase support of polyamide/Kieselguhr resin (2.0 g). The
amino acids used in the synthesis of the peptide analogs are
Fmoc-aa-Opfp derivatives
(9-Fluorenylmethylcarbonyl-aa-O-pentafluorophenyl ester) of each
amino acid(aa) involved in the considered sequences using 0.8 mol
of each amino acid to sequentially form the peptide.
[0042] Each cycle of synthesis may be performed at room temperature
(20.degree. C.) and involves the following steps of reaction:
[0043] Step 1--Deprotection
[0044] The first aa Fmoc-protected at the amino group, was treated
with a 20% solution of piperidine for 7 minutes in order to remove
the Fmoc alpha-protecting group. Washing with dimethylformamide
followed for 12 minutes to remove all traces of piperidine.
Deprotection and washing were run continuously through the column
containing the resin by means of a pump at a flow of 5 ml/min.
[0045] Step 2--Activation of the Fmoc-aa-Opfp derivative
[0046] The amino and carboxy-protected amino acid due, according to
the desired sequence, was activated after its dissolution in 5 ml
of dimethylformamide, by a catalytic amount of hydroxybenzotriazol
(0.5 ml of a 5% w/v solution in dimethylformamide).
[0047] Step 3--Acylation
[0048] The activated and protected Fmoc-aa-Opfp derivative was then
recycled for 30 minutes through the column by the pump at 5ml/min
in order to obtain coupling of the introduced aa at the alpha-amino
group (previously deprotected as reported in Step 1) of the amino
acid preceding the new one in the desired sequence.
[0049] Step 4--Washing
[0050] Washing of the matrix in the column followed by
dimethylformamide for 2 minutes at 5 ml/min before a new cycle
began.
[0051] At the completion of the synthesis, the peptide on the resin
support was cleaved by 95% Trifluoroacetic acid (TFA) with 5%
ethane dithiol as a scavenger, if Cysteine residues were present in
the aa sequence, at room temperature for 2 hours. After separation
of the cleaved peptide from the resin by filtration, the solution
was concentrated by vacuum evaporation to dryness. The collected
solid residue was then solubilized in 10% acetic acid at a
concentration of 10-20 mg/ml and several extractions by diethyl
ether followed (six to eight extractions with half the volume of
the peptide solution) in order to remove the scavenger Ethane
dithiol. The peptide solution was then neutralized by 0.1 N
ammonium hydroxide and adjusted to the concentration of roughly 0.1
mg/ml. The solution was then stirred under air for 1 to 6 hours in
order to obtain the selective oxidation of the two sulfhydryl
groups belonging to the Cys residues of the sequence. In this way,
only monomeric oxidized peptides were obtained with no traces of
polymeric material. The solution of oxidized peptide was then
desalted by reverse-phase chromatography on SEP-PAK C-18 cartridges
(MILLIPORE) and finally freeze dried. The products were analyzed by
high-performance liquid chromatography (HPLC) analysis as well as
by chemical analysis of the synthetic structures.
[0052] Fast atom bombardment may be used to confirm the calculated
mass of the peptides.
[0053] The vaccines may be administered parenterally, preferably
subcutaneously using well known pharmaceutical carriers or inert
diluents such as water for injection, sterile normal saline and the
like.
[0054] The LPS and the peptide may be reacted by combining sterile
aqueous solutions of the LPS and the peptide followed by incubation
for 15 min. to six hours at temperature from 25.degree. C. to
40.degree. C. Generally, the effective amount of the endotoxoid
complex is from 0.1 .mu.g to 50 .mu.g/kg of body weight for a
mammal. The endotoxoid complex may be employed in humans and in
veterinary practice to prevent sepsis and the toxic effects of
endotoxin related shock caused by bacterial infections wherein the
causative organism elaborates endotoxin. The endotoxoid complex may
be administered prophylactically as a vaccine by giving one or more
doses to a subject until a protective level of antibodies is
detected by the following test: Enzyme Linked Immunoassay (ELISA)
or any other clinically acceptable immunoassay.
[0055] Generally, a vaccination regimen may comprise an initial
dose of the vaccine followed by from one to four booster
inoculations given at intervals of two to four weeks.
[0056] The particular dose of a particular endotoxoid complex may
be varied within or without the range that is specified herein
depending on the particular host. Those who are skilled in the art
may ascertain the proper dose using standard procedures. The
vaccine of the invention may be monovalent in that it contains one
endotoxoid complex derived from the LPS obtained from one species
of bacteria or it may be polyvalent and contain a plurality of
endotoxoid complexes made from LPS which is obtained different
species of bacteria. The endotoxoid complex may also be
administered as a part of a multicomponent vaccine such as
diphtheria-pertussis-tetanus (DPT) (Tri-immunol, Lederle
Laboratories) or diphtheria-pertussis-tetanus-haemophilius)
(Tetraimmune, Lederle Laboratories). In such cases, an effective
amount of the endotoxoid complex or complexes may be combined with
the multicomponent vaccine in order to simultaneously induce
multiple antibodies in a host.
[0057] The invention also includes the combined administration of
the vaccine of the invention with an effective amount of an
antibiotic and/ or a peptide as described above to simultaneously
treat a gram-negative infection and provide an immunizing dose of
the vaccine. The amounts of the peptide to be administered are
described in U.S. 5,589,459 and the combined antibiotic-peptide
therapy is described in Serial No 08/456,112, both of which are
incorporated by reference.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
EXAMPLE
[0058] Preparation of Antigens
[0059] LPS from N. meningitidis A1, BB431, 44/77 and purified N.
meningitidis LPS A1 were prepared and characterized according to
the method of Gu and Tsai, Infect. Immun. (1993); 61:1873-1879,
which is incorporated by reference. The cyclic peptide, described
herein by as SAEP2 was synthesized on solid phase, oxidized and
characterized according to the methods set forth in Science (1993):
259:361-365, which is incorporated by reference.
[0060] The covalent conjugate BSA-LPS A1 was prepared as follows:
LPS A1 (5 mg/ml) in PBS., containing two reactive moles of amino
group per mole of oligosaccharide-lipid A monomer, was transformed
to the mono-succinimidyl ester by incubation (60min at room
temperature) with the bis-succinimidyl ester of adipic acid (0.7
mg/ml) in dimethylsulfoxide (DMSO), according to the procedure
reported for amino-activated bacterial capsular oligosaccharides in
Mol Immunol (1985):22:907-919.
[0061] The LPS A1 derivative had more than 98% of the amino groups
contained in its structure transformed into highly reactive ester
groups as determined by the trinitrobenzene sulfonic acid (TNBS)
reaction. The ester-derivative of LPS A1 was then mixed with a
sodium bicarbonate solution pH=8.0 containing 0.9 mg/ml of BSA. The
stoichiometry of the reagents is equivalent to a molar ratio
between the amino groups of BSA:monoester groups of LPS A1=2. The
solution was stirred for 4 hours at room temperature and the
BSA-LPS A1 conjugate was recovered by precipitation with ethanol
(60% v/v final concentration), solubilized in 0.1 M sodium
bicarbonate and finally purified using gel chromatography
(Sepharose, Pharmacia) sterile filtered using a 0.22 .mu.m membrane
and freeze dried. The conjugate is consistent with a ratio of
BSA:LPS A1=1(w/w).
[0062] Preparation of Vaccines
[0063] Complexes of the SAEP2 peptide with both LPS A1 and BSA-LPS
A1 conjugate were prepared by combining sterile solutions of LPS A1
or BSA:LPS A1 with a 0.1 to 5% (w/w) solution of SAEP2 peptide at a
ratio of LPS/SAEP2 peptide of 1:250(w/w). The solutions were
incubated for 30 min. at 37.degree. C. Sodium merthiolate 0.01%
(w/v) was added as a preservative and the products were stored at
4.degree. C. The immunizing dose of LPS A1 was in the range of
0.5-5 .mu.g in 0.2 ml, in the formulations of vaccine tested.
[0064] Testing
[0065] The safety of the LPS A1/SAEP2 peptide complex was confirmed
"in vitro" by the lack of pyrogenic activity tested by Lipid
A-induced LAL clotting and "in vivo" by the determination of TNF
(the mediator of toxicity) titers induced by the vaccines in mice
(strains SW and CD1). TNF was inhibited by 90% when the LPS
A1/SAEP2 peptide complex (1:250w/w ratio) was employed and TNF was
inhibited by 98% when the conjugate BSA-LPS A1/SAEP2 peptide
complex (1:250w/w ratio) was employed. The results for the various
TNF determinations are shown in FIG. 1.
[0066] The immunogenicity of the LPS A1/SAEP2 peptide complex was
determined by comparing the kinetic pattern of the IgG antibodies
specific for LPS A1 which are induced by LPS A1; LPS A1/SAEP2
peptide complex; and BSA-LPS A1; and BSA-LPS A1/SAEP2 peptide
complex. The results are shown in FIG. 2a and FIG. 2b. The test
data show that immunogenicity of endotoxin A1 (LPS A1/SAEP2 peptide
complex) is comparable to that induced by the conjugates BSA-LPS A1
peptide complex. Therefore, toxicity and immunogenicity have been
dissociated in an endotoxoid.
EXAMPLE 2
[0067] LPS from N. meningitidis group A (LPS A1) and Salmonella
enterica (serotype typhimurium, LPS Ty) were used to prepare
vaccines from two groups of structurally different LPS (R- and
S-like chemotype) that originated from clinical isolates of
extracellular and intracellular Gram-negative bacteria,
respectively. Both LPS' exhibit an active lipid A moiety with a
binding site which is stoichiometrically saturated "in vitro", with
high affinity, by a synthetic cyclic peptide (SAEP2) which
exemplifies the peptides of the invention ) For "in vivo" testing,
peptide complexes were prepared at a ratio of (a) 1:250; 1:1000 and
1:2500 of LPS A1:peptide and LPS Ty:peptide.
[0068] The two endotoxoids (A1 and Ty) were completely non-toxic
with respect to LPS, as demonstrated by the level of TNF
systemically released in mice after four injections of the antigens
(FIG. 3). Comparable results were obtained in rabbits by using a
hemorrhagic necrosis test or Schwartzman reaction.
[0069] Outbred mice were immunized subcutaneously, three weeks
apart, by plain LPS A1 and LPS Ty in parallel with the homologous
endotoxoids prepared by complex formation with a cyclic peptide
(SAEP2 was used as an example). Serum immune response was assayed
for specific anti-LPS IgG isotype antibodies, two weeks following
each administration. LPS A1 and LPS Ty have shown a minimum
immunogenic activity at the dose of 5 ug/mouse. The homologous
endotoxoids have expressed, at a dose ten times lower (0.5 .mu.g),
an immunogenic activity comparable to that obtained with a dose of
5 .mu.g of plain LPS.
[0070] To explain this observation, it is hypothesized that there
is a downregulating activity of TNF on T-cells. For this purpose,
mice were immunized with plain LPS A1, whose toxicity was abrogated
by the previous administration of a characterized anti-TNF
monoclonal antibody. Although the toxicity of plain LPS A1 was
abrogated by the anti-TNF treatment (FIG. 3), no significant
increase in the immunogenicity of LPS was detected, in contrast to
the homologous endotoxoid (FIG. 4a), suggesting that while TNF is
the recognized mediator of LPS toxicity, it is not significantly
involved in the immunogenic activity of LPS.
[0071] The endotoxoid-induced IgG antibodies were specific for the
core oligosaccharide chain of LPS A1 and for the O-saccharide chain
of LPS Ty respectively (FIG. 5a and FIG. 5b). The endotoxoids were
functional in fixing and activating homologous and heterologous
species of complement and were completely protective in a
significant model of salmonella infection (FIG. 6a and FIG. 6b).
Furthermore, the endotoxoid-induced IgG antibodies were able to
passively protect the animals from the endotoxemic effects,
detectable by serum TNF release, of a systemic challenge by
homologous LPS (Table I).
Table I
[0072] Effect of anti LPS A1 polyclonal IgG antibodies on the
inhibition of serum TNF production in CD! mice challenged i.v. with
homologous N. meningitidis A1 LPS and heterologous E. coli 055 B5
LPS. Data represent mean of two independent experiments with 5
mice/group. IgG anti LPS A1 were injected i.v. 30 minutes before
either N. meningitidis A1 LPS or E. coli 055:B5 LPS i.v.
challenge.
3TABLE I Effect of anti LPS A1 polyclonal IgG antibodies on the
inhibition of serum TNF production in CD! mice challenged i.v. with
homologous N. meningitidis A1 LPS and heterologous E. coli 055 B5
LPS. Data represent mean of two independent experiments with 5
mice/group. IgG anti LPS A1 were injected i.v. 30 minutes before
either N. meningitidis A1 LPS or E. coli 055:B5 LPS i.v. challenge.
CHALLENGE Mice IgG antiLPS A1 LPS A1 LPS B5 10 saline 6,110+/-2,062
2,371+/-1,471 10 0.25 1,579+/-591 2,068+/-1,864 (inhibition 74%, p
< 0.01)
[0073] These experimental results show the protective activity, in
a mammalian host, of a bacterial endotoxoid originating from LPS of
either extracellular or intracellular gram-negative pathogens.
[0074] While certain preferred and alternative embodiments of the
invention have been set forth for purposes of disclosing the
invention, modifications to the disclosed embodiments may occur to
those who are skilled in the art. Accordingly, the appended claims
are intended to cover all embodiments of the invention and
modifications thereof which do not depart from the spirit and scope
of the invention.
Sequence CWU 0
0
* * * * *